The objective of this project is to characterize the mechanisms of inhibition of DNA replication by the carcinogen 7Beta, 8Alpha-dihydroxy-9Alpha, 10Alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE-I) in cultured human fibroblasts. Effects on initiation of replicons, elongation and joining of nascent DNA strands to high molecular weight, and RNA synthesis will be evaluated in BPDE-I treated cells. By using mutant cell strains exhibiting deficiencies in excision repair or replicative by-pass the modifying effects of these processes will also be examined. Velocity sedimentation analyses will be used to describe the size distribution and quantity of pulse-labeled nascent DNA after BPDE-I treatment. In this technique, an inhibition of replicon initiation is manifested by an initial and selective inhibition of synthesis of nascent DNA with the same length as an average replicon (20 Mum); with time this inhibition spreads to include even larger molecules. Inhibition of DNA chain elongation is manifested by a uniform and dose-dependent reduction in synthesis of DNA in replicons that were already in operation at the time of carcinogen treatment. The relationship between this inhibition and the concurrent synthesis of abnormally small nascent molecules will be investigated using S1 nuclease as a probe for single-stranded regions opposite gaps in daughter DNA. These S1 nuclease-sensitive regions will be detected by the formation of small duplex DNA molecules which contain nascent strands synthesized after BPDE-I damage of the template DNA. Using aphidicolin to synchronize cells at the beginning of the S phase, the growth rates of nascent DNA will be determined in control and BPDE-I-treated cells. The synchronization should produce a less complex distribution of nascent DNA in the alkaline sucrose gradients facilitating analysis of its transition to higher molecular weights. A possible indirect effect of BPDE-I on replication resulting from inhibition of RNA transcription will be investigated by comparing the effects of known inhibitors of RNA synthesis on replicon initiation and DNA chain elongation. These studies should further clarify whether BPDE-I exerts its critical effects in a manner similar to ultraviolet radiation and help to establish the generality of a specific pattern of effects of carcinogens on DNA replication.